Voltage-gated calcium channels (VGCC) play an integral role in the regulation of membrane ion conductance, neurotransmitter release, and cellular excitability. VGCC are composed of the pore-forming α1 subunit and auxiliary α2δ and β subunits that modulate channel expression and functional properties (Dolphin, A. C. British Journal of Pharmacology 2006, 147 (Suppl. 1), S56-S62). These channels can be classified into low-voltage activated (LVA; T-type or Cav3.x) and high-voltage activated (HVA; L-type or Cav1.x and N-, P/Q- and R-types or Cav2.x) channels. N-, P/Q and R channels typically activate at more positive membrane potentials (˜−30 mV) and are involved in “presynaptic” neurotransmission (McGivern J. G. Drug Discovery Today 2006, 11, 245-253). T-type channels are activated at relatively negative membrane potentials (˜−60 mV) and are primarily involved in “postsynaptic” excitability (Shin, H.-S.; et al. Curr. Opin. in Pharmacology 2008, 8, 33-41).
N-type channel α1 subunits are encoded by a single gene (α1B or Cav2.2) in contrast to pharmacologically defined L- and T-type currents that are encoded by multiple α1-subunit genes. A diversity of N-type channels arises due to extensive alternative splicing of the α subunit gene that generates variants with different expression patterns and GPCR-modulated biophysical properties (Gray, A. C.; et al. Cell Calcium, 2007, 42(4-5), 409-417). The primary sequence for Cav2.2 is highly conserved across species (rat and human share 91% identity at the amino acid level).
N-type channels are widely expressed in the central nervous system (CNS) (cortex, hippocampus, striatum, thalamus, brain stem nuclei and spinal cord) and in the peripheral nervous system (PNS) (adult sympathetic nervous system and dorsal root ganglia) (Ino, M.; et al. Proc. Natl. Acad. Sci. USA 2001, 98(9), 5323-5328). In pain pathways, N-type channels are expressed in the rostral ventral medulla, an important site of descending pain modulation (Urban, M. O.; et al. Neuroreport 2005, 16(6), 563-566) and are a major contributor to the synaptic neurotransmission that occurs between C/Aδ nociceptors and spinal lamina I neurons (Bao, J.; et al. J. Neurosci. 1998, 18(21), 8740-50. Heinke, B.; et al. Eur. J. Neurosci. 2004, 19(1), 103-111). In contrast, P/Q type channels are expressed almost exclusively in laminae II-IV of the spinal cord and show little co-localization with Substance P and N-type channels (Westenbroek, R. E.; et al. J. Neurosci. 1998, 18(16), 6319-6330).
Following nerve injury there is increased expression of Cav2.2 (Westenbroek, R. E.; et al. J. Neurosci. 1998, 18(16), 6319-6330. Cizkova, D.; et al. Exp. Brain Res. 2002, 147, 456-463. Yokoyama, K.; et al. Anesthesiology 2003, 99(6), 1364-1370) and α2δ1 subunits (Luo, Z. D.; et al. J. Neurosci. 2001, 21(6), 1868-1875. Newton, R. A.; et al. Mol. Brain. Res. 2001, 95(1-2), 1-8) in addition to increases in the superficial layers of the dorsal horn of the spinal cord supporting a role for N-type channels in neuropathic pain. Recently a nociceptor-specific Cav2.2 splice variant has been identified in the dorsal root ganglion (Bell, T. J.; et al. Neuron 2004, 41(1), 127-138). These channels have distinct electrophysiological properties and current densities (Castiglioni, A. J.; et al. J. Physiol. 2006, 576(Pt 1), 119-134) compared to wildtype Cav2.2 channels. While G-protein coupled receptor inhibition of wildtype N-type channels is typically mediated by Gβγ and is voltage-dependent, the nociceptor specific splice variant is inhibited by GPCR activation (e.g. opioids) in a voltage-independent fashion (Raingo, J.; et al. Nat. Neurosci. 2007, 10(3), 285-292). This mechanism substantially increases the sensitivity of Cav2.2 channels to opiates and gamma-aminobutyric acid (GABA) suggesting that cell-specific alternative splicing of mRNA for Cav2.2 channels serves as a molecular switch that controls the sensitivity of N-type channels to neurotransmitters and drugs that modulate nociception. Collectively these data provide further support for the role of Cav2.2 channels in pain states.
The relative contributions of various HVA Ca2+ channels in nociceptive signaling have been evaluated using knockout mice studies. Cav2.2 knockout mice are healthy, fertile, and do not display overt neurological deficits (Ino, M.; et al. Proc. Natl. Acad. Sci. USA 2001, 98(9), 5323-5328. Kim, C.; et al. Mol. Cell. Neurosci. 2001, 18(2), 235-245. Hatakeyama, S.; et al. Neuroreport 2001, 12(11), 2423-2427. Liu; L.; et al. J. Bioenerg. Biomembr. 2003, 35(6), 671-685). This finding suggests that other types of Cav channels are able to compensate for the lack of Cav2.2 channels at most synapses in these mice (Pietrobon, D. Curr. Opin. Neurobiol. 2005, 15(3), 257-265). Cav2.2 deficient mice are resistant to the development of inflammatory and neuropathic pain (Kim, C.; et al. Mol. Cell. Neurosci. 2001, 18(2), 235-245. Hatakeyama, S.; et al. Neuroreport 2001, 12(11), 2423-2427. Saegusa, H.; et al. EMBO J. 2001, 20(10), 2349-2356), have decreased sympathetic nervous system function (Ino, M.; et al. Proc. Natl. Acad. Sci. USA 2001, 98(9), 5323-5328), and altered responses to both ethanol and anesthetics (Newton, R. A.; et al. Brain Res. Mol. Brain. Res. 2001, 95(1-2), 1-8. Takei, R. et al. Neurosci. Lett. 2003, 350(1), 41-45). Additional behavioral studies indicate that Cav2.2 knockout mice are less anxious, are hyperactive, and show enhanced vigilance compared to wild-type littermates (Beuckmann, C. T.; et al. J. Neurosci. 2003, 23(17), 6793-6797).
N- and P/Q-type channels are localized at neuronal synaptic junctions and contribute significantly to neurotransmitter release (Olivera, B. M.; et al. Annu. Rev. Biochem. 1994, 63, 823-867. Miljanich, G P.; et al. Annu. Rev. Pharmacol. Toxicol. 1995, 35, 707-734). N-type channels play a major role in the release of glutamate, acetylcholine, dopamine, norepinephrine, GABA, substance P and calcitonin gene-related protein (CGRP). P/Q-type channels may be involved in the release of glutamate, aspartate, 5HT, GABA and probably glycine (Pietrobon, D. Curr. Opin. Neurobiol. 2005, 15(3), 257-265).
L, P/Q and N-type channels are blocked by channel specific antagonists i.e., dihydropyridines, ω-agatoxin WA and ω-conotoxin MVIIA/ziconotide, respectively. Agatoxin IVa has been shown to block excitatory (Luebke, J. I.; et al. Neuron 1993, 11(5), 895-902) as well as inhibitory neurotransmission (Takahashi, T.; et al. Nature 1993, 366(6451), 156-158). Intrathecal injection of selective N-type channel blockers (e.g. conotoxin-derived peptides such as GVIA, MVIIA (ziconotide), and CVID) significantly attenuates pain responses in animal models of neuropathic pain, formalin-induced pain, and post-operative pain (Chaplan, S. R.; et al. J. Pharmacol. Exp. Ther. 1994, 269(3), 1117-1123. Malmberg, A. B.; et al. J. Neurosci. 1994, 14(8), 4882-4890. Bowersox, S. S.; et al. J. Pharmacol. Exp. Ther. 1996, 279(3), 1243-1249. Wang, Y. X.; et al. Pain 2000, 84(2-3), 151-158. Scott, D. A.; et al. Eur. J. Pharmacol. 2002, 451(3), 279-286). These peptide blockers bind to the pore region of the channel, do not show voltage- or frequency-dependent activity, and show irreversible channel block (Feng, Z. P.; et al. J. Biol. Chem. 2003, 278(22), 20171-20178). Ziconotide potently blocks neurotransmitter release in the spinal cord dorsal horn (Matthews, E. A.; et al. Pain 2001, 92(1-2), 235-246. Smith, M. T.; et al. Pain 2002, 96(1-2), 119-127. Heinke, B.; et al. Eur. J. Neurosci. 2004, 19(1), 103-111) and in dorsal root ganglion (DRG) neurons (Evans, A. R.; et al. Brain Res. 1996, 712(2), 265-273. Smith, M. T.; et al. Pain 2002, 96(1-2), 119-127). It also potently and fully blocks depolarization-induced release of substance P from rat spinal cord slices. In contrast, intrathecal delivery of the selective P/Q type blocker ω-agatoxin IVA had no effects on mechanical allodynia in the spinal nerve ligation model (Chaplan, S. R.; et al. J. Pharmacol. Exp. Ther. 1994, 269(3), 1117-1123) or thermal hyperalgesia in the chronic constriction injury model (Yamamoto, T.; et al. Brain Res. 1998, 794(2), 329-332) of neuropathic pain.
T-Type or LVA calcium channels are composed of a single pore forming α1 subunit of which there are three subtypes: Cav3.1, Cav3.2 and Cav3.3 (Perez-Reyes, E.; et al. J. Pharmacol. Exp. Ther. 2009, 328(2), 621-7). These channels are activated at relatively hyperpolarized cell membrane potentials and contribute to membrane depolarization following action potential generation. As a result, T-type calcium channel activation triggers secondary bursts of neuronal action potentials with increased action potential duration. Evidence supporting a role of T-type calcium channels in neuropathic pain comes from studies that have shown a concurrent increase in the expression of Cav3.2 channels after-depolarization potentials in medium diameter Aδ high threshold mechanoreceptor dorsal root ganglia (DRG) neurons in diabetic neuropathy (Jagodic, M. M.; et al. J Neurosci 2007, 27, 3305-3316) and in small diameter neurons from the chronic constriction injury (CCl) neuropathic pain model (Jagodic, M. M.; et al. J Neurophysiol 2008, 99, 3151-3156). Additional support comes from gene knockdown studies whereby intrathecal Cav3.2 antisense administration produces a significant knockdown (˜80-90%) of T-type calcium currents in small and medium diameter DRG neurons, and produces robust anti-allodynic and antihyperalgesic effects in the CCl rat model of neuropathic pain (Bourinet, E.; et al. Embo J 2005, 24, 315-324). Moreover, Cav3.2 knockout mice show decreased pain responses compared to wild-type mice in acute mechanical, thermal, and chemical pain models (Choi, S.; et al. Genes Brain Behav 2007, 6, 425-431).
Recently, T-type calcium channel blockers have been proposed to have potential in treating schizophrenia and substance dependence. The T-type calcium channels are located in brain regions that have relevance to schizophrenia and substance dependence (Talley, E. M.; et al. J Neurosci 1999, 19, 1895-1911). More importantly, it has been demonstrated that selective T-type calcium channel blockers, such as TTA-A2, have antipsychotic-like effects in preclinical animal models of psychosis (Uslaner, J. M.; et al. Neuropharmacology 2012, 62, 1413-1421) and were able to decrease nicotine seeking behavior in rats trained to self-administer nicotine (Uslaner, J. M.; et al. Biol Psychiatry 2010, 68, 712-718).
In addition to a role in nociception, T-type calcium channels have also been implicated to play roles in sleep disorders and absence epilepsy (Shin, H.-S.; et al. Curr Opin Pharmacol, 2008, 8, 33-41). Based on expression in the thalamus, T-type calcium channels may play a role in arousal from sleep (Benington, J. H.; et al. Prog Neurobiol 2003, 69, 71-101; Nordskog, B. K.; et al. Neuroscience 2006, 141, 1365-1373). Expression in the adrenal, pituitary and pineal glands suggests that these channels modulate hormone secretion. Notably, Cav3.2 knockout mice appear normal and healthy, although smaller than wild-type mice (Chen, C.-C.; et al. Science 2003, 302, 1416-1418; Choi, S.; et al. Genes Brain Behav 2007, 6, 425-431).
Pain is the most common symptom of disease and the most frequent complaint with which patients present to physicians. Inadequate pain management across the spectrum of pain etiologies remains a major public health problem. Going forward, the development of novel therapeutics with new mechanisms of action for the treatment of pain including calcium channel blockade will have a significant impact on the ongoing struggle to balance efficacy and safety for those patients most in need. The compounds of the present invention are novel calcium channel blockers that have utility in treating pain, amongst other conditions.